To evaluate the feasibility of developing serodiagnostic tests for detecting MAC infections, we have examined the humoral response to MAC disease of HIV-seronegative and AIDS patients. Antibodies to MAC antigens were measured by ELISA and immunoblot analyses in sera from 20 AIDS patients with disseminated MAC disease, 5 HIV-seronegative patients with pulmonary MAC infections, and 20 healthy controls. ELISA studies using MAC sonic extracts demonstrated that the healthy controls and and the AIDS patients with MAC disease had comparable low level anti-MAC antibody titers. The antibody titers from patients with MAC disease who were HIV- negative were significantly higher. However, immunoblot analyses using MAC sonic extracts indicated that each group exhibited a limited heterogeneous response to M. avium antigens. No significant immunoblot reactivity differences were detected. Immunoblot studies using purified nontuberculous mycobacterial antigens revealed that patient sera frequently recognized recombinant proteins derived from 19-, 27-, and 35 kDa mycobacterial antigens. Anti-19 and 27 kDa antibodies were identified in about 50% of MAC patient sera. Greater than 90% of patient sera and only 25% of controls reacted with MK35. These results conclusively demonstrate that the 19-, 27-, and 35 kDa proteins are serologically active MAC antigens. In addition, although sonic extracts do not permit adequate serological discrimination between patient and control sera, recombinant antigens such as MK35 may be useful as serological indicators of nontuberculous mycobacterial disease.